Activators that enhance the initiation of transcription by RNA polymerase B (II) are composed of at least two functional domains: a DNA binding domain and an activating domain (M. Ptashne, Nature 335:683-689 (1988); P. J. Mitchell et al., Science 245:371-378 (1989)). These two domains are generally separable functional units and each can actually be interchanged with the complementary region of an unrelated activator, thereby creating functional chimeric activators (S. Green et al., Nature 325:75-78 (1987)).
A number of structure-function analyses of eukaryotic transcriptional activators have been performed, focussing primarily on the yeast GAL4 and GCN4 proteins and on members of the nuclear receptor family. GAL4 and GCN4 proteins activate transcription by binding to specific upstream activation sequence, which have many of the characteristics of higher eukaryotic enhancer elements (K. Struhl, Cell 49:295-297 (1987)). The herpes simplex activator VP16 represents another type of activator, which activates transcription by binding to the DNA-bound octamer transcription factor rather than binding to the DNA directly (T. Gerster et al., Proc. Natl. Acad. Sci. USA 85:6347-6351 (1988)).
The nuclear receptor family, which includes receptors for steroid hormones, thyroid hormones, vitamin D, and the vitamin A derivative retinoic acid, are also transcriptional enhancer factors which bind DNA directly in the presence of their cognate ligand by recognition of specific enhancer elements, i.e., hormone- or ligand-responsive elements (R. M. Evans, Cell 240:889-895 (1988)). These cognate ligands tend to be small, hydrophobic molecules, including steroid hormones such as estrogen and progesterone, thyroid hormone, vitamin D, and various retinoids (S. Halachmi et al., Science 264:1455-1458 (1994); Gronemeyer, H. and Laudet, V., Protein Profile 2:1173-1308 (1995)).
Despite their small size and apparently simple structure, however, the cognate ligands associated with NRs are known to elicit a wide range of physiological responses. Adrenal steroids for example, such as cortisol and aldosterone, widely influence body homeostasis, controlling glycogen and mineral metabolism, have widespread effects on the immune and nervous systems, and influence the growth and differentiation of cultured cells. The sex hormones (progesterone, estrogen and testosterone) provoke the development and determination of the embryonic reproductive system, masculinize/feminize the brain at birth, control reproduction and related behavior in adults and are responsible for development of secondary sex characteristics. Vitamin D is necessary for proper bone development and plays a critical role in calcium metabolism and bone differentiation. Significantly, aberrant production of these hormones has been associated with a broad spectrum of clinical disease, including cancer and similar pathologic conditions.
All NRs display a modular structure, with five to six distinct regions, termed A-F. The N-terminal A/B region contains the activation function AF-1, which can activate transcription constitutively. Region C encompasses the DNA binding domain (DBD), which recognizes cognate cis-acting elements. Region E contains the ligand-binding domain (LBD), a dimerization surface and the ligand-dependent transcriptional activation function AF-2 (reviewed in Mangelsdorft, D. J. et al., Cell 83:835-839 (1995a); Mangelsdorft & Evans, Cell 83:841-850 (1995b); Beato, M. et al., Cell 83:851-857 (1995); Gronemeyer & Laudet, “Transcription Factors 3: Nuclear Receptors”, in Protein Profile, vol. 2, Academic Press (1995); Kastner, P. et al, EMBO J. 11:629-642 (1992); Chambon, P., FASEB J 10:940-954 (1996)).
Several classes of domains in activators are capable of mediating transcriptional activation. Yeast activators GAL4 and GCN4 and herpes simplex VP16 all contain activation domains that are composed of acidic stretches of amino acids, which may act by forming amphipathic a helices (I. A. Hope et al., Cell 46:885-894 (1986); J. Ma et al, Cell 48:847-853 (1987); E. Giniger et al., Nature 330:670-672 (1987); S. J. Triezenberg et al, Genes Dev. 2:718-729 (1988)). The activation functions of human Sp1 and CTF/NFI proteins contain glutamine- and proline-rich areas, respectively (A. J. Courey et al., Cell 55:887-898 (1988); N. Mermod et al., Cell 58:741-753 (1989)). Studies with steroid hormone receptors have shown that both the N-terminal A/B domain and the C-terminal hormone binding domain (RBD) contain transcription activation functions (AFs) (M. T. Bocquel et al., Nucl. Acids Res., 17:2581-2595 (1989); L. Tora et al, Cell 59:477487 (1989)). The AFs of the human estrogen receptor (hER) do not contain stretches of acidic amino acids (S. Halachmi et al., Science 264:1455-1458 (1994)). Conversely, however, the human glucocorticoid receptor (hGR) contains two activation functions, τ-1 (located in the A/B domain) and τ-2 (located in the N-terminal region of the HBD), both of which are acidic (S. M. Hollenberg et al., Cell 55:899-906 (1988)).
From the results of studies on transcriptional interference/squelching between nuclear receptors and on homo- and heterosynergistic stimulation of initiation of transcription from minimal promoters by the activation functions present in hER (AF-1 and AF-2) and the acidic activator VP16, it has been proposed that AFs may activate transcription by interacting with different components of the basic initiation complex (Bocquel et al., Nucl. Acids. Res. 17:2581-2595 (1989); Meyer et al., Cell 57:433-442 (1989); L. Tora et al., Cell 59:477487 (1989)). Studies of the transcriptional interference/squelching properties of AADs, hER AF-1 and hER AF-2, however, showed that both hER AF-1 and AF-2 can squelch acidic activators, such as VP 16, but that the converse was not true, i.e., AADs do not squelch hER AF-1 or AF-2. Moreover, hER AF-1 and AF-2, which are clearly distinguished by their synergistic properties, nevertheless squelch each other (D. Tasset et al., Cell 62:1177-1187 (1990)).
Based on these results, it was proposed that a string of transcriptional intermediary factors (TIFs) exists, interposed between enhancer factors and the basic transcriptional factors. For example, AF-1 and AF-2 have been suggested to contact the string of TIFs at functionally equivalent points, while AADs are believed to interact at an earlier point in the series (D. Tasset et al., Cell 62:1177-1187 (1990)).
Several putative coactivator TIFs for NR AF-2s have been characterized (see Chambon, P., FASEB J 10:940-954 (1996); Glass, C. K. et al., Current Opin. Cell Biol. 9:222-232 (1997); Horwitz, K. B. et al., Mol. Endocrinol. 10:1167-1177 (1996) for recent reviews). In particular, LeDouarin, B. et al., EMBO J. 15:6701-6715 (1996) have demonstrated that a 10-amino acid fragment of TIF1α is necessary and sufficient to mediate interaction with RXR in a ligand- and AF-2 integrity-dependent manner. Notably, within this TIF1α fragment, they identified a LxxLLL (SEQ ID NO: 13) motif, termed NR box, whose integrity is required for interaction with nuclear receptors, and pointed out that this motif is conserved in several other putative coactivators (LeDouarin, B. et al, EMBO J. 15:6701-6715 (1996)) Whereas TIF1α and several other putative coactivators do not, or only very poorly, stimulate transactivation by NRs in transiently transfected mammalian cels, the TIF2/SRC-1 family (Onate, S. A. et al., Science 270:1354-1357 (1995); Voegel, J. J. et al., EMBO J. 15:3667-3675 (1996)), the CBP/p300 family (Kamei, Y. et al., Cell 85:403414 (1996), Chakravarti D. et al, Nature 5:99-103 (1996); Hanstein, B., et al., Proc. Natl. Acad Sci USA 93:11540-11545 (1996); Smith, C. L. et al., Proc. Natl. Acad. Sci. USA 93:8884-8888 (1996); for recent reviews see Eckner, R., Biol. Chem. 377:685-688 (1996); Janknecht & Hunter, Current Biol. 6:951-954 (1996b); Shikama, N. et al., Trends in Cell Biol. 7:230-236 (1997)) and the androgen receptor coactivator ARA70 (Yeh & Chang, Proc. Natl. Acad. Sci. USA 93:5517-5521 (1996)) have been unequivocally shown to enhance AF-2 activity.
In addition to binding NRs, CBP/p300 can also interact directly with SRC-1 (Kamei, Y. et al., Cell 85:403-414 (1996); Yao, T. P. et al., Proc. Natl. Acad. Sci. USA 93:10626-10631 (1996)) and both factors have been shown to exert histone acetyltransferase activity (Bannister & Kouzarides, Nature 384:641-643 (1996); Ogryzko, V. V. et al., Cell 87:953-959 (1996)). Moreover, CBP/p300 can recruit p/CAF which is itself a nuclear histone acetyltransferase (Yang, X. J. et al., Nature 382:319-324 (1996)). However, apart from interacting with coactivators in a ligand-dependent manner, NRs have also been shown to interact, often in a ligand-independent fashion, directly or indirectly with components of the transcriptional machinery, suds as TFIIB, TBP, TAFs, or TFIIH (Baniahmad et al., (1993)); Jacq, X. et al., Cell 79:107-117 (1994); Schulman, IG. et al., Mol. Cell Biol. 16:3807-3813 (1996); May, M et al., EMBO J. 15:3093-3104 (1996); Mengus, G. et al., Genes & Dev. 11: 1381-1395 (1997)).
Hong, H. et al., Proc. Natl. Acad. Sci. USA 93:4948-4952 (1996) originally described a partial cDNA of the mouse homologue of TIF2, named GRIP1, and recently reported the isolation of a full length GRIP1 cDNA (Hong, H. et al., Mol. Cell. Biol. 17:2735-2744 (1997)). Using the yeast Saccharomyces cerevisiae as a model system, they have shown that transcriptional activation by TR, RAR and RXR, could also be stimulated by GRIP1 coexpression, which suggests that TIF2/GRIP1 could be a general coactivator for NRs (Hong, H. et al., Mol. Cell. Biol. 17:2735-2744 (1997)).
The overall picture emerging from multiple recent studies on the mechanisms by which nuclear receptors modulate target gene transcription involves three subsequent steps, (i) the ligand-induced transconformation of the NR LBD, which results in (ii) the dissociation of corepressors and formation of TIFs/coactivator complexes, which themselves (iii) through interaction with additional downstream factors (e.g., CBP, p300) modulate the acetylation status of core histones and, thus, chromatin condensation/decondensation. Histone acetylation on its own is, however, insufficient for transcription activation (Wong et al, (1997)), and a simultaneous or subsequent fourth event comprises the direct and/or indirect recruitment of elements of the transcription machinery (e.g., TFIB, TBP, TAFs, TFIIH; Jacq, X. et al., Cell 79:107-117 (1994); Schulman, IG. et al., Mol. Cell. Biol. 16:3807-3813 (1996); May, M. et al., EMBO J. 15:3093-3104 (1996); Mengus, G. et al., Genes & Dev. 11: 1381-1395 (1997)). Note that such interactions do not need to be ligand-dependent, if the primary function of the liganded LBD (AF-2) is to regulate DNA accessibility through chromatin remodeling. Indeed, several of the reported interactions between NRs and general transcription factors occur in a ligand-independent manner. Accordingly, there is a need in the art for the isolation and characterization of transcriptional intermediary factors.